Substrate affinity of photosensitizers derived from chlorophyll-a: the ABCG2 transporter affects the phototoxic response of side population stem cell-like cancer cells to photodynamic therapy

Abstract

Photosensitizers (PS) synthesized with the aim of optimizing photodynamic therapy (PDT) of tumors do not always fulfill their potential when tested in vitro and in vivo in different tumor models. The ATP-dependent transporter ABCG2, a multidrug resistant pump expressed at variable levels in cancerous cells, can bind and efflux a wide range of structurally different classes of compounds including several PS used preclinically and clinically such as porphyrins and chlorins. ABCG2 may lower intracellular levels of substrate PS below the threshold for cell death in tumors treated by PDT, leaving resistant cells to repopulate the tumor. To determine some of the structural factors that affect substrate affinity of PS for ABCG2, we used an ABCG2-expressing cell line (HEK 293 482R) and its nonexpressing counterpart, and tyrosine kinase ABCG2 inhibitors in a simple flow cytometric assay to identify PS effluxed by the ABCG2 pump. We tested a series of conjugates of substrate PS with different groups attached at different positions on the tetrapyrrole macrocycle to examine whether a change in affinity for the pump occurred and whether such changes depended on the position or the structure/type of the attached group. PS without substitutions including pyropheophorbides and purpurinimides were generally substrates for ABCG2, but carbohydrate groups conjugated at positions 8, 12, 13, and 17 but not at position 3 abrogated ABCG2 affinity regardless of structure or linking moiety. At position 3, affinity was retained with the addition of iodobenzene, alkyl chains and monosaccharides, but not with disaccharides. This suggests that structural characteristics at position 3 may offer important contributions to requirements for binding to ABCG2. We examined several tumor cell lines for ABCG2 activity, and found that although some cell lines had negligible ABCG2 activity in bulk, they contained a small ABCG2-expressing side population (SP) thought to contain cells which are responsible for initiating tumor regrowth. We examined the relevance of the SP to PDT resistance with ABCG2 substrates in vitro and in vivo in the murine mammary tumor 4T1. We show for the first time in vivo that the substrate PS HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) but not the nonsubstrate PS HPPH-Gal (a galactose conjugate of HPPH) selectively preserved the SP which was primarily responsible for regrowth in vitro. The SP could be targeted by addition of imatinib mesylate, a tyrosine kinase inhibitor which inhibits the ATPase activity of ABCG2, and prevents efflux of substrates. A PDT resistant SP may be responsible for recurrences observed both preclinically and clinically. To prevent ABCG2 mediated resistance, choosing nonsubstrate PS or administering an ABCG2 inhibitor alongside a substrate PS might be advantageous when treating ABCG2-expressing tumors with PDT.

Figure 1

The effect mediated by the TKI IM on fluorescence produced by photosensitizers related to pyropheophorbide-a (PhA) with different groups attached to the macrocycle, as indicated in Chart 1. Bars are mean+/− SEM of 2–13 experiments with triplicate samples for each compound. HEK 293 482R cells (ABCG2 +) (■), HEK 293 PcDNA cells (ABCG2−) (■). * P<0.05.

Figure 2

Carbohydrate substitutions on HPPH. The effect mediated by the TKI IM on fluorescence produced by HPPH with different groups attached to the macrocycle, as indicated in Chart 2. Bars are mean+/− SEM of 2–3 experiments with triplicate samples for each compound. HEK-293 482R cells (■), HEK-293 PcDNA cells (■).

Figure 3

The effect mediated by the TKI IM on fluorescence produced by purpurinimides (15 and 22) with lactose attached at different positions of the macrocycle (16–21), and glucose (23) or galactose (24) attached at position 3 as indicated in Charts 3 and 4. Bars are mean+/− SEM of 2–5 experiments with triplicate samples for each compound. HEK-293 482R cells (■), HEK-293 PcDNA cells (■).* P<0.05.

Figure 4

Flow cytometry dot plots with red fluorescence on the x-axis and blue fluorescence on the y axis, showing the side population (SP) in 4T1 cells grown in vitro and stained with DCV. The numbers in the plots show the SP as a percentage of the single live cells in the gated population, with DCV alone, control, left: Con, or incubated with the inhibitors FTC and IM. The SP and NON-SP gates in the control show the population from which cells were sorted for comparative phototoxicity with HPPH-PDT.

Figure 5

A: The SP remaining in 4T1 cells treated in vitro with HPPH or HPPH-Gal PDT in the presence and absence of IM. Cells were incubated for 4 h with 800 nM HPPH or HPPH-Gal, in the presence or absence of 10 μM IM and irradiated with 1 J/cm² then harvested and stained with DCV 48 h later. Bars are mean and SD of 3 experiments. * Significantly different from dark groups P<0.05. B: Dose response curves of HPPH-PDT of sorted SP and NON-SP (from populations indicated in Figure 4) and unsorted 4T1 cells treated in vitro with HPPH PDT. Cells were incubated for 4 h with HPPH and irradiated with 1 J/cm² then the surviving fraction was determined by the colorimetric MTT assay 48 h later. Mean and SD of 3 experiments: sorted SP (●); sorted SP + IM (❍); sorted NON-SP (■); unsorted cells (◆); combined phototoxicity data from sorted SP and NON-SP (▲), determined by mathematical combination of sorted SP and NON-SP surviving fractions: [1 - (non surviving fraction NON-SP - non surviving fraction SP)].

Figure 6

The SP in cells from 4T1 tumors treated in vivo with HPPH or HPPH-Gal PDT (0.47 μmol/kg i.v. and irradiated 24 h later with 665 nm laser light, 135 J/cm² at 75 mW/cm²). Tumors were harvested 48 h later, disaggregated and stained with DCV and 7-AAD to determine the SP of surviving cells by flow cytometry. Bars are the accumulated means and SD of 4–5 samples from 2 experiments containing 1–3 pooled tumors in each sample. * Significantly different from untreated group, P<0.05. Tumors were pooled to obtain sufficient cells for a statistically valid analysis, since some of the tumors were very responsive to treatment, and few cells were recovered.

Figure 7

A: Relative change in HPPH fluorescence of several cell lines after incubation in the absence and presence of 10 μM IM. The black portion of the columns indicated the relative change above the level of fluorescence found by incubating with HPPH alone. * indicates a significant change in HPPH fluorescence mediated by IM (p<0.05). B: SP present in cell lines in which the relative change in HPPH fluorescence after incubation in the presence of 10 μM IM was not significantly different from incubation with HPPH alone.

Figure 8

ABCG2 distribution after immunostaining with BXP-21 antibody to human ABCG2. A: HEK-293 R482A; B: HEK-293 PcDNA; C: HeLa (ABCG2+); D: HeLa (ABCG2−); E: U87; F: FaDu; G: A549; H:A549 (Isotype control). Short arrows: membrane ABCG2; long arrow: cytoplasmic ABCG2; white arrow: nuclear ABCG2.

Chart 1

Photosensitizers related to pyropheophorbide-a. ^aCompounds 1 and 2 are commonly known as pyropheophorbide-a (PPA) and HPPH respectively. Compound 4 is 3-(1'-m-iodobenzyloxy) pyropheophorbide-a. ^b Reference. ^c Reference. ^d Reference

Chart 2

Carbohydrate analogs of HPPH (2-(1’-hexyloxyethyl) pyropheophorbide-a) linked at position 17 with a flexible amide bond or a rigid amino-amide linker. ^a Reference

Chart 3

Lactose analogues of N-substituted mesopurpurinimide (15). ^a References,

Chart 4

Carbohydrate analogues of N-hexyl-purpurinimide (18) and 3-hydroxymethyl-N-hexyl-purpurinimide (22). ^a Reference